Immunogenic cancer peptides and uses thereof

ABSTRACT

This invention relates to novel general methods and compositions that provide cancer-specific or highly cancer-associated antigens useful for diagnosis and treatment of cancer.

This invention relates to novel general methods and compositions thatprovide cancer-specific or highly cancer-associated antigens useful fordiagnosis and treatment of cancer.

BACKGROUND

The utilization of cancer-specific antigens and molecular markers in thediagnosis and treatment of malignant tumors is a goal of medicalprofessionals. The realization of this goal has been advanced by the useof in vivo animal and in-vitro model systems in order to map out therelevant steps of a cancer-specific immune response and also the stepsrequired for its use in cancer therapy. Methods which utilizecancer-specific and/or cancer-associated markers for diagnosis andtherapy have been reported, but the principal shortcoming preventing theimplementation of these methods has been the paucity of cancer-specificor highly cancer-associated antigens and other markers of cancer inhumans.

Some progress in obtaining candidates for cancer-specific or highlycancer associated antigens for cancer diagnosis and treatment includesthe construction of synthetic peptides, for example, for the productionof antibodies specific for the peptides, where the peptides arepotentially useful as markers. For example, different epitopes have beenfound to be associated with mucins from malignant cells, in contrast tomucins in non-malignant cells. Aberrant glycosylation has been found insome peptides from tumors.

Vaccines and immunotherapies using specific domains of membrane proteinshave been reported to be more effective than vaccines and immunotherapyusing entire glycoproteins.

At present, not enough cancer antigens or markers are available for usein implementing robust cancer diagnostic or therapeutic methods inhumans. Human cancer antigens and markers described to date are eitherinadequate or too few in number to provide useful clinical tools.

For example, the MAGE family of antigens described by Boon et al. (1994)are reported to be cancer-associated antigens. Cancer-associatedantigens are those expressed in greater quantity in molecules in or on,or derived from cancer cells, but are also concurrently expressed inmolecules from normal cells. This duality complicates therapeuticutility of the antigens for vaccines and antibodies where positiveeffects are dependent upon reaching a therapeutic dose before a toxicdose level is realized. Other limitations of the MAGE antigens are thatthey are also intracellular cancer antigens thus greatly diminishingtheir utility for cancer cell targeting which is more effective for cellsurface antigens. Intracellular antigens serve as poor localizingtargets for immunotherapy, targeted cytotoxic therapeutic agents, cellreceptor blocking agents, other cell-surface disruptive agents, and fordiagnostic imaging. They are poor immunogenic targets for eliciting ameasurable immune response. Their release for direct quantification isunpredictable because cancer cell disruption is required.

Cheever et al.(1997a,b) have described the potential diagnostic andtherapeutic use of oncogenic proteins which are expressed by both cancerand normal cells. They describe using oncogenic proteins withsite-specific mutations as the cancer-specific antigens. However, theoncogenic proteins cited by Cheever, designated the p21 proteins, areintracellular and thus share the drawbacks of other intracellularantigens, that is, cannot the detected on cell surfaces. Furthermore,mutated expression is not always manifested by expressed oncogenicproteins in all cancer cells, thus leaving some cells to expressoncogenic proteins which are subject to self-recognition and are thuspoorly immunogenic.

Cheever's other example, the erbB-2 epidermal growth factor receptor,also known as HER-2/neu, is used to support the hypothesis that breakingself-recognition offers a novel therapeutic pathway (Disis et al.,1998a, b; 1999) although that method is not commonly accepted by mostimmunologists. The erbB-2 molecule is a transmembrane receptor with asignificant extracellular portion. Its extracellular domain is commonlybelieved to be structurally similar for both cancer cells and normalcells. Thus, the advantages it possesses over intracellular antigencandidates is minimized because of its susceptibility to down regulationof any specific immune response on the basis of self recognition.

Use of derivatives of bombesin, an amphibian protein, was an attempt toinhibit growth of tumor cells that respond to bombesin (Knight et al.,1997). Bogden and Moreau attempted to treat human cancer byadministering analogs of a biologically active peptide to a patient.However, these attempts used molecules that did not differentiate normalfrom cancer cells.

The deglycosylated mucins described by Barratt et al., 1998 andHenderson et al., 1998 are another example of a class ofcancer-associated antigens with epitopes detectable outside of the cell.Mucins are large secreted and/or transmembrane glycoproteins withgreater than 50% of their molecular weight derived from O-linkedcarbohydrates attached to serine and theonine. Their cancer specificitydepends on a greater degree of altered structure rather than onnumerical over-expression. The loss or diminution of carbohydrate sidechains emanating from a central core protein makes the Muc proteins moreimmunogenic. Finn et al. ascribes this immunogenity as a result ofsignificant altered molecular folding made possible by a release frommolecular rigidity conferred by the many projecting glycoside chainsfound in mucin molecules in non-cancerous cells. The alteration infolding creates neo-epitopes which help break immune self-recognitionand also separately facilitates stimulation of a cellular immuneresponse. Problems with the Muc antigens include insufficient diversityneeded to provide wide enough antigenic coverage for many cancers, andtheir rapid cellular release rate as a consequence of Muc antigens beingsecreted proteins, as opposed to functional cell membrane proteins suchas receptor molecules, receptor-like molecules, or cell adhesionmolecules. The latter attribute makes Muc antigens less effectivetherapeutic and imaging targets.

Hudziak et al. (1998a, b) describes the therapeutic utility ofmonoclonal antibodies specific for the extracellular domain of thenormal HER-2/neu receptor (also known as erbB-2). The basis of thistherapeutic method is described as the inhibition of thecancer-proliferative function of the receptor caused by the binding of aspecific monoclonal antibody to the outer domain of the receptor therebypreventing the binding of circulating epidermal growth factor and otherligands to the receptor. Decreased or absent growth factor stimulationresults in cancer cell death through apoptosis. This method relies onhigher expression of Her-2/neu on cancer cells as compared to normalcells. Therapy is dose dependent. Sufficient blocking antibody must beadministered so as to block enough cancer cell HER-2/neu moleculesrequired to affect cancer cell death without causing normal cell deathor normal cell toxicity. Adequate therapeutic dosing is not possible forall patients who express HER-2/neu on the their tumor cells. Some cancerpatients express adequate amounts of HER-2/neu; some express lowamounts; and yet others express none. Consequently, this therapeuticmethod works marginally, or not at all for most patients. Occasionally,when patient circumstances are appropriate, this method is capable ofaffecting total cancer remission. This limited result illustrates thebasic soundness of a therapeutic method provided that a large repertoireof cancer-specific or cancer-associated functional targets were madeavailable. However, more and better cancer-specific andcancer-associated antigens are needed to make these approachesclinically useful.

A method of preparing phosphorylated tumor specific peptides wasreported by Calenoff (1998).

There are suggestions of expression of cancer-specific orcancer-associated molecules, as well as over-expression orunder-expression of the molecules in or on cancer cells. For example,many receptor-like adhesion proteins found on the surface of cells havebeen described. Some of these adhesion proteins are reported tofacilitate tumor migration and invasion (Zheng et al., 1999; Rabinovitzet al., 1995; Friedl et al., 1998) or metastatic spread (Romanov et al.,1999). Others are reported to facilitate essential functioning for bothcancer cells and tissues and for normal cells and tissues (Ekblom et al,1998; Fleischmajer et al., 1998; Bonkoff, 1998; Fujiwara et al., 1998;Lohi, 1998). Blocking certain functions facilitated by receptor-likeadhesion molecules is suggested to provide new therapeutic modalitiesfor eradicating or controlling cancer (Ruoslahti et al., 1997). Althoughvarious adhesion molecule isotypes are reported to be over-expressed(Damiano et al., 1999; Liapis et al., 1996; Begum et al., 1995; Katsuraet al., 1998) or underexpressed (Furakawa et al., 1994; Damjanovich etal., 1997; Luguki et al., 1999) on cancer cells as compared to normalcells, none have been described which possess the cancer-specific orhighly cancer-associated structural modifications of the presentinvention.

SUMMARY OF THE INVENTION

The invention relates to general methods and compositions that providecancer-specific or highly cancer associated antigens useful for cancerdiagnosis and treatment. An aspect of the invention is algorithms fordetermining, selecting and/or constructing synthetic peptides that arecandidates for producing a cancer-specific or cancer-associated immuneresponse useful in the diagnosis and treatment of cancer.

The invention also relates peptides selected by the methods of thepresent invention. The peptides are preferably small, e.g. from 3 toabout 1000 amino acids in length, and are centered around amino acidsthat are generally glycosylated in non-cancerous cells, and are on thecell surface, but are not glycosylated in cancer cells. More preferredlengths of the peptides are from 3-7 amino acids or 3-10, or 5-10,although peptides up to about 25 or to 1000 amino acids in length, arealso within the scope of the invention. The peptides are alsohydrophilic. The peptides or fragments thereof include any variation inthe amino acid sequence, whether by conservative amino acidsubstitution, deletion, or other processes, provided that thepolypeptides are in accord with the criteria of the present invention.More specifically, more than one peptide, the sequences of which are inaccord with the criteria of the present invention, are preferablypresent to enhance the discriminatory power of the immunoassays andtherapies disclosed herein. That is, a plurality of antigenic peptidesforms an array (or repertoire) of molecules suitable for diagnosis andtreatment of cancer.

A peptide of the present invention contains both unmodified and modifiedamino acids. It is recognized that the conversion of a normal to acancerous cell type likely involves many steps. At some point, a cell(more precisely, a group of cells—for example, a tumor) becomesdistinguishable as a “cancer cell”. If at that point, an amino aciddiffers in its state from that in non-cancerous cells, it is definedherein as “modified.” Not all the cells in a cancerous tissuenecessarily have the modification. For purposes of the presentinvention, it suffices that the modification allows some cancer cells tobe distinguished from normal cells by detection of the modification ormodifications.

On the external domain of proteins of cells with normal growth patterns,asparagine is the most frequent site of glycosylation, but in cancercells the peptides of the present invention are missing a glycosidiccomplex altogether. The absence of the glycosidic complex is expected toconfer a cancer-specific or highly cancer-associated immunogenicity tothe altered peptide region. Deglycosylation is expected to remove sterichindrance present in non-cancerous cells, to phosphorylation or othermodifications of the neighboring amino acids. Removal of sterichindrance allows available phosphorylases to add phosphate groups toamino acids usually under the glycosidic umbrella. Addition of phosphategroups facilitated by deglycosylation provides an additionalcancer-specific or cancer-associated molecular structure to be detected.

The immunogenic peptides of the present invention may include one ormore of the constituent amino acids that are chemically modified, eitherin the natural state of the cancer cells, or synthetic, and the chemicalmodification confers upon the peptide a cancer-specific or highlycancer-associated immunogenicity or structured uniqueness that isdifferent from, and may be independent of, the specificity orassociation related to the altered (deglycosylated) glycosylation sites.

Following the steps outlined in Table 1, peptides suitable for thepractice of the invention result in peptides with the formulas shown inTable 2. TABLE 1 Steps in Obtaining Cancer Specific of Cancer-AssociatedAntigenic Peptides Step 1: obtain amino acid sequence of theextracellular domain of a candidate molecule e.g. a receptor orreceptor-like molecule. Step 2: map hydrophilic regions of the domain byanalyzing the amino acid sequence of the domain of step 1 employing therolling sum analysis of 7 consecutive residues. Step 3: identify thehydrophilic regions of step 2 that are glycosylated in non-cancerous(normal) cells, but are deglycosylated in cancer cells. Thedeglycosylated regions of the peptide are candidates for beingcancer-specific or cancer associated peptide antigens. Step 4: look foramino acids to either side of the deglycosylated amino acids identifiedin step 3 that are susceptible to alteration in the absence of sterichinderance by glycoside chains. Step 5: synthesize candidate peptidesthat fit the criteria obtained in steps 3 or 4 and label the peptides atone end e.g. with biotin. Step 6: use synthesized peptides as sourceantigens in immunoassays used to measure peptide-specific antibody inbiological fluids (i.e. serum) from cancer patients and biologicalfluids from control subjects. Peptides which specifically complex withantibody in cancer patient fluids but not in control fluids arecancer-specific antigens. Peptides which complex with antibodies incancer patient fluids more frequently than the complex with antibodiesfrom control fluids from (non cancerous patient, or at least not know tobe cancer patients) are designated cancer-associated antigens. Peptideswhich complex with antibody in both cancer patient fluids and alsocontrol fluids, or with neither, are neither cancer-specific norcancer-associated.

The invention is generally directed to immunogenic peptides whichinclude a sequence of three or more amino acids, possess a nethydrophilic character, and contain at least one amino acid that isglycosylated in normal cells (generally an asparagine residue) butdeglycosylated in cancer cells. As can be seen for the general forms inTable 2, the deglycosylated amino acid is located no further than:

-   -   1. 3 unmodified amino acids away from a fourth unmodified amino        acid on either side of the deglycosylated amino acid;    -   2. 3 amino acids away from the most distal modified amino acid        found on either side of the deglycosylated amino acid, where        distal refers to a location from a deglycosylated amino acid;    -   3. 6 amino acids away from another deglycosylated amino acid (if        there are no modified amino acids in between the two adjacent        deglycosylated amino acids).        Arrays include differentiating pluralities of peptides of the        present invention, to diagnose cancer.

An aspect of the invention is immunoassays employing immunogenicpeptides to measure specific peptide-reactive antibodies in biologicalfluids, more specifically: an aspect of the invention is monoclonalantibodies and antibody-like molecules such as Fab2 and FAb fragments,known to those skilled in the art, and recombinant proteins thereof,which are specifically reactive with the immunogenic peptides of thepresent invention. Immunoassays employing these antibodies orantibody-like molecules of the present invention are used to measure inbiological fluids, molecules containing altered peptide regions whichcorrespond in vivo to the immunogenic peptides of the present invention.

Cancer imaging reagents are developed using labeled molecules of thepresent invention including antibodies or antibody-like molecules,directed toward cancer specific or cancer-associated peptides of thepresent invention. Suitable labels include radioisotopes, a paramagneticlabel, and a water density label. The labels complexed with theantibodies or antibody-like molecules target cancer cells and tissuesand respond to image detectors to identify the location of the cancer.

A therapeutic vaccine containing one or more immunogenic peptides of thepresent invention, and prepared by methods known to those skilled invaccine development, is an aspect of the invention. Adjuvent/peptideconjugates including the immunogenic peptides coupled to molecules whichfacilitate enhanced immunogenicity, are used to stimulate the hostimmune system to facilitate the killing of cancer cells and thereaftermaintain immune surveillance in case of cancer recurrence.

Vaccines created by recombinant techniques containing immunogenicpeptides together with adjuvant molecular sequences which promoteincreased immunogenicity of the immunogenic peptides to stimulate thehost immune system to facilitate the killing of cancer cells andthereafter maintain immune surveillance in case of cancer recurrence,are also within the scope of the invention.

Definitions

The term “antigen presenting cell” (APC) includes “professional antigenpresenting cells” that constitutively express MHC class II molecules(e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells, andactivated T cells in humans) as well as other antigen presenting cellsthat are capable of presenting antigen to T cells. APCs can express theappropriate combination of MHC molecules and costimulatory and/oradhesion molecules known in the art to be sufficient for presentation ofantigen to T cells or can be induced or engineered to express suchmolecules.

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses that are influenced bymodulation of T cell costimulation. Exemplary immune responses include Tcell responses, e.g., proliferation, cytokine production, and cellularcytotoxicity. In addition, the term “immune response” includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages.

“Unmodified amino acids” are those found in the non-cancerous state—thatis, as the amino acids exist in normal cells i.e. non-cancerous cells.Modified amino acids are those that exist in altered states in cancerouscells.

The term “markers,” as used herein, includes any molecule which isdetectable in a biological sample and indicates the presence of anothermolecule of interest. Some markers are antigenic. Markers are usefulbecause their presence is associated with a disease or condition ofinterest. Markers of interest herein are those whose presence isassociated with cancer.

The single letter code for amino acids, well known to those of skill inthe art, is used herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the amino acid sequence (SEQ ID NO: 67) of the humanepidermal growth factor receptor (EGFR); larger letters depictextracellular portions of human epidermal growth factor receptor (EGFR);the bold N denotes normally glycosylated asparagine residues on the EGFRextracellular portions; underlined amino acid sequences=hydrophilicpeptide regions on extracellular portion of EGFR.

FIG. 2 presents amino acid sequence (portions of SEQ ID NO: 67) positionnumbers that indicate regions of cancer-specific/highlycancer-associated immunogenic peptides from the sequence shown in FIG.1; N depicts deglycosylated asparagine in cancer-specific or highlycancer-associated immunogenic peptide regions; an underlined S, T or Yrespectively depicts serine, threonine, and tyrosine amino acids whichcan become aberrantly phosphorylated because absent polysaccharidecomplexes emanating from the highlighted asparagines no longersterically prevent various phosphorylases from approachingphosphorylateable amino acids and attaching a phosphate group; theaddition of a phosphate group creates a novel immunogenic peptide regioncentered by the phosphorylated amino acid(s) as well as thedeglycosylated asparagine(s).

FIG. 3(a) graphically indicates screening results using the EGFR peptide(portion of SEQ ID NO: 67) rNvs; the x-axis shows results for 2 groupsof serum samples:

-   -   a. from patients with squamous cell carcinoma (dark circles);    -   b. samples from patients not known to have any cancer (open        circles);        a dotted line shows the control (non-cancerous) population mean        background+2.5 standard deviations antibody levels (serum IgG)        shown on the y-axis were below the mean background+2.5 standard        deviations of the mean (+2.5 SD) for all samples from persons        not known to have cancer, whereas 2 of 45 of the samples from        persons with squamous cell carcinoma, had antibody levels above        the same mean+2.5 SD; this indicates that this peptide region of        the epidermal growth factor receptor erbB-1, in altered form,        likely serves as a cancer-specific immunogen or target.

FIG. 3(b) graphically indicates screening results using the EGFR peptide(portion of SEQ ID NO: 67) rNvSrgr; the x-axis shows results for 2groups of serum samples:

-   -   a. samples from patients with squamous cell carcinoma (dark        circles);    -   b. samples from patients not known to have any cancer (open        circles);        antibody levels (serum IgG) shown on the y-axis) were below the        mean background+2.5 SD for the serum samples from persons not        known to have any cancer, whereas 3 of 45 serum antibody levels        were above the same mean+2.5 SD for the serum samples for        persons with squamous cell carcinoma; although the peptide        antigen used to elicit these results is structurally related to        the peptide (portion of SEQ ID NO: 67) rNvsr, the serum antibody        levels elicited for the peptide (portion of SEQ ID NO: 67)        rNvSrgr are much higher thus indicating that adding an        aberrantly phosphorylated extension offers a neoantigen which        complexes with specific serum antibody in excess to that        afforded by the (portion of SEQ ID NO: 67) rNvsr peptide alone;        this, too, indicates that this peptide region of the epidermal        growth factor receptor erbB-1, in a second altered form, likely        serves as a cancer-specific immunogen or target.

FIG. 4 graphically indicates screening results using the TROP1 peptidewith the amino acid sequence (SEQ ID NO: 68) aemNgSk; the x-axis shows 2groups of results:

-   -   a. serum samples from persons with squamous cell cancer (dark        circles); and    -   b. serum samples from persons not known to have cancer (open        circles);        the y-axis shows IgG antibody levels; 6 of 45 sera from cancer        patients were above the mean background level from the        controls+2.5 SD, whereas only one serum from the control        population was above that level; this indicates that this        peptide region of the TROP1 cell surface molecule in altered        form, likely serves as a highly cancer-associated immunogen or        target; the single positive result within the control population        may also be indicative of silent (clinically undetectable)        cancer presence in the affected subject.

FIG. 5 graphically illustrates serum antibody levels obtained with aplurality of 4 biotinylated peptides used as test antigens; the x-axisshows 2 groups:

-   -   a. samples from persons with squamous cell cancer (dark        circles);    -   b. samples from persons not known to have cancer (open circles);        the y-axis shows IgG antibody levels; 11 of 45 sera from cancer        patients were above the control mean background level+2.5 SD,        whereas only one serum from the control population was above        that level; this graph illustrates the positive summative effect        of using a sufficiently large number of non-homologous synthetic        peptides corresponding to the humorally antigenic peptide        regions of cancer cell receptors and/or receptor-like molecules;        by having enough suitable antigenic peptides in the antigen mix        of the described immunoassay method, a point is reached where        enough antigenic peptides are available to provide the        immunoassay with a sensitivity approaching 100 percent while        maintaining high specificity.

FIG. 6 graphically illustrates serum antibody levels obtained with 9biotinylated peptides used as test antigens; the x-axis shows 7 groups:

-   -   a. samples from persons with Stage I prostate cancer (dark        circles);    -   b. samples from persons with Stage II prostate cancer (open        circles);    -   c. samples from persons with Stage III prostate cancer (dark        squares);    -   d. samples from persons with Stage IV prostate cancer (open        squares);    -   e. samples from persons with benign prostatic hypertrophy (BPH),        a non-malignant enlargement of the prostate (diamonds with        crosses inside);    -   f. samples from men not known to have cancer or BPH (open        diamonds);    -   g. samples from women not known to have cancer (dark triangles).        The y-axis shows IgG antibody levels; 4 of 7 sera (57%) from        Stage I prostate cancer patients were above the mean background        level+2.5SD; 3 of 7 sera (43%) from Stage II prostate cancer        patients were above that level; 2 of 3 sera (67%) from Stage III        prostate cancer patients were above that level; and 0 of 1 sera        (0%) from Stage IV prostate cancer patients were above that        level; whereas, only one serum (3.6%) from the BPH population        and none of the normal males or females were above the threshold        level; this graph also illustrates the positive summative effect        of using a sufficiently large number of non-homologous synthetic        peptides corresponding to the humorally antigenic peptide        regions of cancer cell receptors and/or receptor-like molecules.

FIG. 7 is a diagram showing a glycosylated (CHO) amino acid (darkcircle) in a peptide (chain of circles) in a non-cancerous cell and anouter membrane protein of a receptor—or receptor like molecule (OMP)with a transmembrane region (dashed line through a cell membrane (CM))attached to an inner cell portion (small dotted circle).

FIG. 8 is a diagram of the same structure as in FIG. 7 with theexception that the glycosylated amino acid in FIG. 7 (dark circle) isnow deglycosylated, as in a cancerous cell.

FIG. 9 is a diagram of the same of structure as in FIG. 8 except thatone of the amino acids (shaded circles) in the peptide is phosphorylated(P), another modification in a cancer cell in addition to thedeglycosylation shown in FIG. 8.

DESCRIPTION OF THE INVENTION

The invention relates to methods and compositions for obtaining cancerspecific or cancer associated antigens (generally antigenic peptides)for use in diagnosis and treatment of cancer. An aspect of the inventionis algorithms for determining, selecting and/or constructing antigensthat are suitable for use in diagnostic tests for cancer, for producingcancer-specific or cancer-associated antibodies for use in diagnosis ortreatment of cancer, and for producing immunogenic constructs fortreatment of cancer. Aspects of the invention include a large repertoire(array) of cancer-specific and cancer-associated peptide antigenslocated on the surface of externally expressed cellular receptors orreceptor-like molecules.

An algorithm for a peptide of the present invention directs among otherthings, that in the amino acid sequence, no more than 3 unmodified aminoacids are located on either side of a modified amino acid (see Tables 1and 2). Amino acid modification may include phosphorylation, conjugationof oxidized radicals (Tsimikas et al., 1999; Brame et al., 1999), and/orconjugation of glycosides(Shamsi et al., 1998) which differ from theglycosides which are normally attached to the peptide.

The antigens of the present invention generally have the followingattributes and characteristics:

-   -   a. the antigens possess cancer-specific or cancer-associated        alterations which confer antigenic and/or structural specificity        upon the extracellular domains of commonly expressed receptor or        receptor-like molecules;    -   b. the receptors and receptor-like molecules are potentially        expressed by all cancer types thereby providing broad-based        antigenic diversity and significant quantitative expression for        most cancers;    -   c. the receptors and receptor-like molecules can serve as cell        surface cancer-specific or cancer-associated antigens or as cell        surface cancer markers;    -   d. the receptors and receptor-like molecules are found as early        as Stage I as well as in Stages II, III, and IV of cancer        progression;    -   f. because the receptor and receptor-like repertoire is        significantly large, diminished expression among different        cancers of some receptor or receptor-like molecules of the array        is compensated by the standard expression or over-expression of        other receptors or receptor-like molecules of the repertoire        thereby providing sufficient antigenic and/or marker coverage.        This varying molecular expression allows diagnostic        discrimination of individual cancer types;    -   g. because the receptor and receptor-like repertoire is        significantly large, enough receptor or receptor-like molecules        are available which either remain affixed to the outer surface        of cancer cells thereby serving as ideal antigenic or marker        targets for diagnosis or therapy, or are predictably released        into the peripheral circulation or biological fluids which bath        the cancer cells thereby serving as shed cancer antigens to be        measured for diagnostic purposes.

The antigen repertoire of this invention is different from the antigensreported by others in the following ways:

-   -   a. the MAGE antigens of Boon (1994) expressed in cancer cells        are not structurally unique compared to MAGE concomitantly        expressed in normal cells. MAGE antigens are intracellularly        expressed and therefore require cancer cell damage or        fragmentation for reliable extracellular expression;    -   b. the antigens described by Cheever (1997) possess        cancer-specific structural alterations but are intracellular, or        are over-expressed on the outer surface of cancer cells, but        lack cancer-specific structural alterations which would confer        immunogenic and/or marker specificity;    -   c. the Muc antigens described by Finn (1998 a, b) possess        cancer-associated structural specificity that confers antigenic        and/or marker specificity but are excreted and thereby poorly        retained on the cancer cell surface. The structural alterations        of Muc antigens are different from the alterations described for        the antigens of the present invention. The antigenic/marker        sites of peptides for this invention are small and are therefore        less affected by a twisting-type of conformational change but        rely more on peptide denuding and upon the modification of amino        acids which are normally hidden by attached glycosides. The Muc        antigen repertoire is numerically insufficient to provide across        the board coverage for adenocarcinomas which express Muc        antigens;

d. the antigenic site employed in the method of Hudziak (1998 a,b) totarget the HER-2/neu epidermal growth factor receptor is described asbeing structurally similar to HER-2/neu expressed by non-cancerouscells. TABLE 2 Examples of formulas for suitable candidate cancerantigenic peptides (where u = unmodified amino acid; N = deglycosylatedamino acid; M = modified amino acid and [ ]n symbolizes n number ofrepeats of a basic unit in brackets) are: uNu uNuu Nuuu uNuuu uuNuuuuNuuu uuuNuuu uNuMuuu uuNuMuuu uuuNuMuuu uuuNMMuuu uuuNuMMuuuuuuNMMMuuu uNuuMuu uNuuMuuu uuNuuMuuu uuuNuuMuuu uuNuuMuuNuuuuuNuuMuuNuuu uuuMuuNuuMuuu [uuNuuN]n [uuuNuuuN]n [uuuNuuuuN]n[uuuNuuuuuN]n [uuuNuuuuuN]n [uuuNuuuuuuN]n [uuuNuuuuuuNu]n[uuuNuuuuuuNuu]n [uuuNuuuuuuNuuu]n [uuMuuN]n

TABLE 3 Comparison of Peptides Present in Serum From Cancer Patients toPeptide Sequences Cancer Tissue (SEQ ID NOS 1-66, respectively, in orderof appearance) Presence of Cell Surface Prostate Cancer Absence of CSPProteins Serum Test in Prostate (CSP) Tested CSP Peptides Result CancerE-Cadherin vkNst (SEQ ID NO: 1) + + N-Cadherin dkNls (SEQ ID NO: 2) + +CD9 nnNNss (SEQ ID NO: 3) − − CD38 dkNst (SEQ ID NO: 4) − − CD40 gtNkt(SEQ ID NO: 5) + + CD44 drNgt (SEQ ID NO: 6) + + NhSeg (SEQ ID NO: 7) +spNhs (SEQ ID NO: 8) − CD46 drNht (SEQ ID NO: 9) − − CD53 sdNst (SEQ IDNO: 10) − − CD55 fcNrs (SEQ ID NO: 11) − CD63 knNht (SEQ ID NO: 12) − −CD66 saNrs (SEQ ID NO: 13) + + trNdt (SEQ ID NO: 14) − skNqs (SEQ ID NO:15) − CD82 pgNrt (SEQ ID NO: 16) − − desmoglein 1 tkNgt (SEQ ID NO: 17)− − desmoglein 2 kiNat (SEQ ID NO: 18) − − erbB-1 daNkt (SEQ ID NO:19) + + perNrt (SEQ ID NO: 20) − crNvs (SEQ ID NO: 21) − erbB-2 dtNrs(SEQ ID NO: 22) + + erbB-3 heNct (SEQ ID NO: 23) − + erbB-4 aeNct (SEQID NO: 24) − − pdNct (SEQ ID NO: 25) − fgfr1 esNrt (SEQ ID NO: 26) − +fgfr2 ekNgs (SEQ ID NO: 27) − + fgfr4 diNss (SEQ ID NO: 28) − −hepatocyte gfr vgNks (SEQ ID NO: 29) − + pdgfr alpha eeNns (SEQ ID NO:30) − + pdgfr beta kdNrt (SEQ ID NO: 31) − − trNvs (SEQ ID NO: 32) −ICAM 1 hkNqt (SEQ ID NO: 33) − + integrin alpha 1 qrNit (SEQ ID NO: 34)− − seNas (SEQ ID NO: 35) − integrin alpha 2 drNhs (SEQ ID NO: 36) − −integrin alpha 3 meNkt (SEQ ID NO: 37) + + leNht (SEQ ID NO: 38) − rmNit(SEQ ID NO: 39) − integrin alpha 5 kaNts (SEQ ID NO: 40) − − lrNes (SEQID NO: 41) − integrin alpha 6 raNhs (SEQ ID NO: 42) − + integrin alpha 9qkNqt (SEQ ID NO: 43) − − kgNcs (SEQ ID NO: 44) integrin alpha (v) qdNkt(SEQ ID NO: 45) + + kaNtt (SEQ ID NO: 46) − teNqt (SEQ ID NO: 47) −ekNdt (SEQ ID NO: 48) − integrin beta I nkNVt (SEQ ID NO: 49) + + vtNrs(SEQ ID NO: 50) − kNvtNrs (SEQ ID NO: 51) − keNss (SEQ ID NO: 52) −eqNct (SEQ ID NO: 53) integrin beta 3 skNfs (SEQ ID NO: 54) − − integrinbeta 5 rcNgs (SEQ ID NO: 55) − − pdNqt (SEQ ID NO: 56) − integrin beta 6qkNss (SEQ ID NO: 57) − − evNss (SEQ ID NO: 58) − protein-tyrosine seNdt(SEQ ID NO: 59) − − phosphatase PCP-2 protein-tyrosine keNdt (SEQ ID NO:60) − − phosphatase kappa ghNes (SEQ ID NO: 61) − gdNrt (SEQ ID NO: 62)− tgfr beta type II deNit (SEQ ID NO: 63) + + eyNts (SEQ ID NO: 64) +insulin-like gf pdNdt (SEQ ID NO: 65) − + receptor rnNtt (SEQ ID NO: 66)−

Table 3 column 2 to the right is a compilation of cancer-modifiedpeptide regions to be found on 41 receptors, receptor-like molecules, oradhesion molecules reported in the literature (CSP). This tableillustrates the diversity of tissue and organ types which possessreceptors, receptor-like molecules, or adhesion molecules able topresent with cancer-specific or highly cancer-associated structuralalterations.

Table 3 third column illustrates the positive or negative reactivitybetween peptides representing modified peptide regions of cancer cellsurface protein molecules with antibodies in sera from prostate cancerpatients. The presence or absence in prostate cancer cells of the 41tested molecules is shown as + or − in column four. Table 3 illustratesthat reactivity exists between most of the modified peptides of prostatecancer cell surface molecules but shows no reactivity with modifiedpeptide regions of molecules not found in prostate cancer, thusillustrating the ability to serologically determine cancer type byhaving first mapped out the peptide antigen repertoire needed toidentify each cancer. These results support feasibility of using thepeptides of the present invention, in particular a plurality ofpeptides, for cancer diagnosis.

An aspect of the invention is monoclonal antibodies and antibody-likemolecules such as Fab2 and FAb fragments, known to those skilled in theart, and recombinant proteins (Hussain et al., 1996).

A cancer imaging reagent is developed using molecules including labeledantibodies or antibody-like molecules directed to antigenic peptides ofthe present invention. Suitable labels include a radioisotopic label forthe cancer imaging reagents which, upon binding to the cells that form acancerous tumor, highlight the presence of the tumor when scanned with anuclear medicine scanner (Goldenberg, 1993; 1999).

Another suitable label is a paramagnetic label which, upon binding tothe cells of a cancerous tumor, highlights the presence of the tumorwhen scanned with a nuclear magnetic resonance (NMR) scanner (To et al.,1992).

Another suitable label comprises a water density label which, uponbinding to the cells of a cancerous tumor, highlights the presence ofthe tumor when scanned with a CAT scanner.

Cancer therapeutic reagents developed using the molecules includingantibodies or antibody-like molecules directed to the peptides of thepresent invention, have at least one of the following characteristics:they (1) bind to a cancer cell and promote lysis of that cell; (2) bindto and block the function of a receptor or receptor-like molecule on acancer cell, thereby promoting a reduction or cessation of cancer cellgrowth or promoting cancer cell death; and (3) carry a radioisotope or atoxin which upon binding to a cancer cell damages or promotes cancercell death (Goldenberg, 1993).

Examples of cancer therapeutic methods which can be formulated using asuitable cancer antigens/markers array are (see Materials and Methodsfor details and citations):

-   -   a. passive immunization using constructs such as engineered        antigen presenting cells and production of antigen presenting        dendritic cells able to stimulate the host immune system to        recognize and kill cancer cells;    -   b. active immunization using cancer vaccines including        recombinant fusion proteins, vaccine compositions containing        adjuvants, vaccine compositions containing nucleic acid        molecules, recombinant microorganisms which express cancer        antigens, antigen/antibody conjugates wherein the antibody acts        as a delivery vehicle for targeting the antigen onto antigen        presenting cells, and heat shock protein/antigen complexes;    -   c. cell lytic therapeutic antibodies, cell adhesion blocking        antibodies, and growth factor receptor blocking antibodies.

Therapeutic methods using the non-phosphorylated peptide antigens of thepresent invention, either their amino acid sequences or thecorresponding nucleic acid sequences that encode the peptides includethe following:

-   -   a. passive immunization using constructs such as engineered        antigen presenting cells and production of antigen presenting        dendritic cells able to stimulate the host immune system to        recognize and kill cancer cells;    -   b. active immunization using cancer vaccines including        recombinant fusion proteins, vaccine compositions containing        adjuvants, vaccine compositions containing nucleic acid        molecules, recombinant microorganisms which express cancer        antigens, antigen/antibody conjugates wherein the antibody acts        as a delivery vehicle for targeting the antigen onto antigen        presenting cells, and heat shock protein/antigen complexes.

Criteria for an antigen array suitable for passive immunotherapyspecific for cancerous cells include the following:

-   -   1. each type of cancer possesses antigens such as protein,        peptide, carbohydrate, or lipid molecules which are structurally        unique as compared to non-cancerous cells and are also        immunogenic;    -   2. the cancer antigens are located on the cell surface so they        are sufficiently accessible for targeting T cells;    -   3. the cancer antigens are present at the earliest stages of        cancer progression as well as during later stages;    -   4. each cancer cell must have on its surface a sufficient number        of specific antigens to serve as an adequate target for an        effective cellular-mediated immune response;    -   5. the cancer antigens are retained on the surface of the cancer        cells for a time sufficient for the therapeutic T cells to find        their target and also to retain the bound T cells for a time        sufficient to affect cancer cell death.

Criteria, for an antigen array suitable for developing effectiveconstructs for active immunotherapy include the following:

-   -   1. each cancer type possesses antigens such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells and are also immunogenic;    -   2. the cancer antigens are located on the cell surface to be        sufficiently accessible and thus more easily recognized by the        host immune system;    -   3. the cancer antigens are present at the earliest stages of        cancer progression as well as during later stages;    -   4. each cancer cell has on its surface a sufficient number of        specific antigens that serve as an adequate target for a humoral        and/or cellular-mediated immune response;    -   5. the cancer antigens are retained on the surface of the cancer        cells for a time sufficient for the therapeutic effector cells,        and antibodies elicited by the immunostimulatory constructs to        find their target and also to retain the effector cells and        antibodies for a time sufficient to affect cancer cell death.

Criteria for a marker array including an antigen suitable for developingcell-lytic therapeutic antibodies include the following:

-   -   1. each cancer type possesses markers such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells;    -   2. the cancer markers are available in sufficient numbers on the        surface of cancer cells to provide an adequate therapeutic        target at the earliest stages of cancer progression as well as        during later stages;    -   3. the cancer markers are retained on the surface of the cancer        cells for a time sufficient for the therapeutic antibodies to        find their target and also to retain the bound antibody for a        time sufficient to affect cancer cell death.

Criteria for a marker array suitable for developing growth factorreceptor blocking antibodies include:

-   -   1. each cancer type possesses markers such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells;    -   2. the cancer markers are available in sufficient numbers on the        surface of cancer cells to provide an adequate therapeutic        target at the earliest stages of cancer progression as well as        at later stages;    -   3. the cancer markers are retained on the surface of the cancer        cells for a time sufficient for the growth factor receptor        blocking antibodies to find their target and also to retain the        bound antibody for a time sufficient to affect cancer cell        death;

Criteria for a marker array suitable for developing cell surfaceadhesion blocking antibodies include:

-   -   1. each cancer type possesses receptor-like adhesion molecules        which are structurally unique as compared to non-cancerous        cells;    -   2. the adhesion molecules are available in sufficient numbers on        the surface of cancer cells to provide an adequate therapeutic        target at the earliest stages of cancer progression as well as        at later stages;    -   3. the cancer markers are retained on the surface of the cancer        cells for a time sufficient for adhesion blocking antibodies to        find their target and also to retain the bound antibody for a        time sufficient to prevent cancer cell attachment, migration,        de-differentiation or other function essential for cancer cell        survival or metastasis.

Those of skill in the art recognize that identification of Stage Icancer generally provides a 90 percent or greater cure rate through theuse of currently available cancer therapies (DeVita et al., 1985).Therefore, diagnostic assays for early stage cancer are extremelyimportant.

Examples of cancer diagnostic methods which can be formulated using asuitable cancer antigen/marker repertoire are: cancer-specific antibodyassays, cancer-specific antigen assays, and in-vivo cancer imaging.

Criteria for an antigen array suitable for developing cancer-specificantibody assays include:

-   -   1. each cancer type possesses antigens such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells and are also immunogenic;    -   2. the cancer antigens are located on the cell surface to be        sufficiently accessible and thus more easily recognized by the        host immune system;    -   3. enough cancer cells have on their surface a sufficient number        of specific antigens to elicit an immune response capable of        being measured at the earliest stages of cancer progression as        well as at later stages and among most affected patients.

Criteria for a marker array suitable for developing cancer-specificantigen-capture immunoassays include the following:

-   -   1. each cancer type possesses markers such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells;    -   2. the cancer markers are predictably secreted or otherwise        released into the pericellular fluids to be reliably measured;    -   3. enough cancer cells shed enough specific marker from within a        cancerous tumor to be reliably measured at the earliest stages        of the tumor's progression and during later stages of most        affected patients.

Criteria for a marker array suitable for developing cancer-specificimaging reagents include the following:

-   -   1. each cancer type possesses markers such as protein, peptide,        carbohydrate, or lipid molecules which are structurally unique        as compared to non-cancerous cells;    -   2. the cancer markers are available in sufficient numbers on the        surface of cancer cells to provide an adequate imaging target at        the earliest stages of cancer progression as well as during        later stages;    -   3. the cancer markers are retained on the surface of the cancer        cells for a time sufficient for the imaging agents to find their        target and also to retain the bound imaging agent for a time        sufficient to record the presence and location of the cancer.        Possible Outcomes for Peptides Screened as Antigens in Serum        Antibody Assays:

1. A positive result indicating the presence of a peptide-specificantibody in cancer patient biological fluid samples, absent evidence ofantibody in samples from subjects without cancer (FIG. 3) indicates thetested peptide is a cancer-specific peptide (immunogen).

2. A significantly higher positive prevalence of a peptide-specificantibody in cancer patient biological fluid samples as compared tosamples from subjects without cancer (FIG. 4) indicates either that thetested peptide is cancer specific and that the few control positiveshave asymptomatic cancer or that the peptide serves as a highlycancer-associated antigen.

3. No difference in positive antibody levels between cancer patients andsubjects without cancer. Biotinylated peptides producing these resultsare neither cancer specific nor highly cancer-associated.

EXAMPLES

The following examples illustrate embodiments of the invention.

Example 1 Use of the EGFR Peptide on Serum from Cancer Patients andControls

Using immunoassay 2 (see Materials and Methods) the following resultswere obtained.

FIG. 3(a) graphically indicates screening results using the EGFR peptide(portion of SEQ ID NO: 67) rNvs; the x-axis shows results for 2 groupsof serum samples:

-   -   a. from patients with squamous cell carcinoma;    -   b. samples from patients not known to have any cancer; antibody        levels (serum IgG) were below the mean background plus 4        standard deviations of the mean (+2.5 SD) for all samples from        persons not known to have cancer, whereas 2 of 45 of the samples        from persons with squamous cell carcinoma, had antibody levels        above the mean+4SD.

FIG. 3(b) graphically indicates screening results using the EGFR peptide(portion of SEQ ID NO: 67) rNvSrgr; the x-axis shows results for 2groups of serum samples:

-   -   a. samples from patients with squamous cell carcinoma;    -   b. samples from patients not known to have any cancer; antibody        levels (serum IgG) were below the median background plus 2.5 SD        for the serum samples from persons not known to have any cancer,        whereas 3 of 45 serum levels were above the median+2.5 SD for        the serum samples for persons with squamous cell carcinoma.

Example 2 Use of the TROP1 Peptide on Serum Samples from Cancer PatientsCompared to Controls

FIG. 4 graphically indicates screening results using the TROP1 peptidewith the amino acid sequence (portion of SEQ ID NO: 68) emNgSk; thex-axis shows 2 groups of results:

-   -   a. serum samples from persons with squamous cell cancer; and    -   b. serum samples from persons not known to have cancer; the        y-axis shows IgG antibody levels; 6 of 45 serum from cancer        patients were above the median background level+2.5 SD, whereas        only one serum from the control population was above that level.        Materials and Methods        A Method for Selecting Cancer-Specific or Highly        Cancer-Associated Immunogenic Peptides and/or Markers

The identification and validation (or confirmation) of cancer-specificand cancer-associated antigenic peptide regions and/or marker peptideregions found on the extracellular domain of receptors or receptor-likemolecules is performed through the use of a algorithms such as thefollowing:.

-   -   First, the amino acid sequence of the extracellular domain of a        receptor or receptor-like molecules is obtained. For example,        the human epidermal growth factor receptor (EGFR), erbB-1, as        illustrated in FIG. 1.    -   Second, the amino acid sequence is analyzed employing rolling        sum analysis of 7 consecutive residues (Hopp et al., 1981;        Parker et al., 1986; Fauchere et al., 1983; Taragu et al., 1990)        in order to map out peptide regions which are hydrophilic and        therefore apt to be expressed on the outer surface of the        protein. For example, the hydrophilic regions of the EGFR outer        domain are underlined in FIG. 1.    -   Third, hydrophilic peptide regions containing amino acids which        are normally glycosylated are identified. These amino acids,        illustrated by a bold capital letter N in FIG. 1, are apt to be        totally (or partially) deglycosylated in cancer cells. The        absence or truncation of the glycoside chain results in peptide        structures which are structurally distinct for cancer cell        proteins. The distinctly structured peptide regions can serve as        a tumor-specific antigenic site if this alteration is not        expressed in normal cells, or can serve as a cancer-associated        antigen by cancer cells. The ability of a peptide to serve as an        antigen depends on the host's immune system being able to        process and recognize the peptide as an antigen. The processing        and recognition of an antigen is dependent on individual MHC        genotypes. If an altered peptide cannot serve as a        cancer-specific or cancer-associated antigen, it may still be        useful as a molecular marker of cancer on the basis of its        cancer-specific or cancer-associated molecular alteration.    -   Fourth, the deglycosylated peptide regions are evaluated for the        inclusive presence of amino acids that are susceptible to        alteration in the absence of glycoside chains which normally        would sterically restrict the contact of enzyme or other agents        with the amino acids susceptible to molecular modification. The        amino acid modification confers a second order alteration on the        affected peptide which can result in a new and distinct peptide        structure with specific antigenic and/or marker properties.        Examples of such an amino acid modification include aberrant        phosphorylation of serine, threonine, and tyrosine residues,        malondialdehyde (MDA) modification of lysine residues, aberrant        glycosylation of arginine residues, and the like.    -   Fifth, candidate peptides which fit the criteria of the        cancer-modified peptide regions described in Steps 1 through 4        are synthesized and biotin labels are attached at either end of        each peptide.    -   Sixth, employing the immunoassays disclosed herein, each        biotinylated peptide is screened as an antigen against sera or        other relevant biological fluids containing antibodies taken        from one of the following groups: cancer patients, patients with        benign lesions or inflammatory conditions, and healthy subjects.        Peptides can also be screened using tumor-infiltrating        lymphocytes or peripheral blood-born lymphocytes from cancer        patients. Candidate peptides we consider cancer-specific or        cancer-associated depend on whether they fit the following        definitions.

Potentially useful peptides prepared in accord with steps 1-5 preferablypossess the following attributes:

-   -   a. contain no more than 3 unmodified amino acids attached on        either side of a deglycosylated amino acid or a modified amino        acid. Peptides containing both deglycosylated amino acids and        modified amino acids have no more than 2 unmodified amino acids        between the deglycosylated amino acids and the modified amino        acids. Peptides with more unmodified amino acids on either side        of a deglycosylated amino acid become antigenically less        differentiating for cancer as the respective unmodified amino        acid numbers increase. The EGFR peptide (portion of SEQ ID        NO: 67) daNktg in FIG. 2 represents a peptide suitable for the        practice of the invention, with a single deglycosylated amino        acid at its central portion. The EGFR peptide (portion of SEQ ID        NO: 67) daNkTglk in FIG. 2 represents a suitable peptide with        both a modified amino acid and a deglycosylated amino acid in        its central portion.    -   b. a plurality of deglycosylated amino acids and modified amino        acids providing that the modified amino acids proximal to a        deglycosylated amino acid are no further than the third amino        acid position from the nearest deglycosylated amino acid and        that 2 or more deglycosylated amino acids in a peptide are        connected by no more than 6 unmodified amino acids.        Synthesis of Deglycosylated Peptides and Phosphorylated,        Modified Forms

Methods of synthesis of peptides and their corresponding, encodingnucleic acid molecules are well know in the art and can be obtainedcommercially from U.S. companies such as the American peptide Company(Sunnyvale, Calif. 94086) and Commonwealth Biotechnologies, Inc.(Richmond, Va. 23235).

Immunoassay Method 1: Used to Detect Serum IgA, IgD, IgE, IgG, and IgMAntibodies Specific for Individual Peptide Antigens

Materials:

Neutravidin coated microtiter plates manufactured as per Example 4.

Wash Buffer: 20 mM Tris-HCl+150 mM NaCl+0.05% Triton X405+0.2 mg/mLthimerosal, pH 7.4.

Biotinylated peptide solution containing 1.5 g/mL peptide in 20 mMTris-HCl+600 mM NaCl+30 mg/mL polyethylene glycol 4000 (PEG-4000,Mallinckrodt Chemical H273-61)+0.05% Triton X405+0.2 mg/mL thimerosal,pH 7.4.

Anti-IgA/alkaline phosphatase (Kirkegaard and Perry075-1001)+anti-IgG/alkaline phosphatase (Kirkegaard and Perry 075-1002)solution: 0.3 g/mL of each conjugate in solution containing 20 mMTris-HCl+600 mM NaCl+30 mg/mL polyethylene glycol 4000 (PEG-4000,Mallinckrodt Chemical H273-61)+0.05% Triton X405+0.2 mg/mL thimerosal,pH 7.4.

4-methylumbelliferyl phosphate (4-MUP) fluorescing substrate solution:

-   -   25.2 mg 4-MUP (Sigma M-8883)/mL solution containing 180 mM        2-amino-2-methyl-1-propanol+123 mM magnesium chloride, pH 9.5.

Fluorolite 1000 microtiter plate fluorometer (Dynatech) with excitationset at 365 nm and emission at 450nm.

Procedure:

1) Adsorb neutravidin (NA) reactive antibodies and biotin reactiveantibodies from serum samples by adding one neutravidin-conjugated paperdisc to every 25 mL of serum and 1 biotin-conjugated paper disc to every200 mL serum. Allow disc/serum mixture to incubate for 26 to 18 hours atroom temperature, under gentle agitation.

2) Mix 75 mL of adsorbed serum together with 75 mL of peptide solution.

3) Vortex mixture and let incubate at room temperature for 40 minutes.

4) Aspirate well contents and wash microtiter wells of neutravidin plate(275 mL wash buffer/well)×6.

5) Add 100 mL biotinylated peptide/serum solution to corresponding welland incubate for 3.5 minutes.

6) Aspirate well contents and wash microtiter wells (275 mL washbuffer/well)×6.

7) Add 100 mL anti-IgA/alkaline phosphatase+anti-IgG/alkalinephosphatase conjugate solution and incubate for 40 minutes.

8) Aspirate well contents and wash microtiter wells (275 mL washbuffer/well)×6.

9) Add 100 mL 4-MUP substrate solution.

10) Read derived fluorescence using microtiter plate fluorometer at 5,10, 20, 30, and 60 minutes.

Immunoassay Method 2: Used to Detect Serum IgA, IgD, IgE, IgG, and IgMAntibodies Specific for Individual Peptide Antigens.

Materials:

NeutrAvidin^(a) conjugated paper disc, 6 mm.

Serum diluent: 10 mM sodium phosphate, pH 7.20, with 150 mM sodiumchloride, and 0.20 mg/mL sodium azide.

NeutrAvidin^(a) coated white microtiter plate, stored in 10 mM Tris-HCl,pH 7.50, containing 600 mM sodium chloride and 0.2 mg/mL thimerosal.

Plate blocking solution: 10 mM sodium phosphate, pH 7.20, containing 150mM sodium chloride, 100 mg/mL Triton X-405, and 0.2 mg/mL thimerosal.

Plate wash buffer: 20 mM Tris chloride, pH 7.4, containing 150 mM sodiumchloride, 0.5 mg/mL Triton X-405 and 0.2 mg/mL thimerosal.

Peptide solution: 0.06 μg/mL peptide dissolved in 20 mM Tris chloride,pH 7.4, containing 600 mM sodium chloride, 30 mg/mL polyethylene glycol4000, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis(§-aminoethyl ether)N,N,N′,N′-tetraacetic acid, 0.5 mg/mLTriton X-405 and 0.2 mg/mL thimerosal.

Control peptide solution: 0.013 μg/mL control peptide dissolved in 20 mMTris chloride, pH 7.4, containing 600 mM sodium chloride, 30 mg/mLpolyethylene glycol 4000, 1 mM ethylenediaminetetraacetic acid, 1 mMethylene glycol-bis(§-aminoethyl ether)N,N,N′,N′-tetraacetic acid, 0.5mg/mL Triton X-405 and 0.2 mg/mL thimerosal.

Conjugate solution: 0.100 μg/mL alkaline phosphatase conjugatedpolyclonal goat anti human IgG dissolved in 20 mM Tris-HCl, pH 7.40,with 600 mM sodium chloride, 30.0 mg/mL PEG4000, 3.0 mg/mL BSA, 0.5mg/mL Triton X-405 and 0.20 mg/mL thimerosal.

Substrate solution: 25.2 μg/mL 4-methylumbelliferyl phosphate dissolvedin 180 mM 2-amino-2-methyl-1-propanol, pH 9.50, containing 123 μMmagnesium chloride.

Serum preparation:

-   -   1. Add 100 μL serum to 15 NeutrAvidin^(a) coated paper discs in        a suitably sized test tube.    -   2. Incubate with gentle mixing at ambient temperature for 16-20        hours.    -   3. Add 7.900 mL of serum diluent and mix gently for 30 minutes.    -   4. Vortex the tube gently to completely release the serum from        the discs.    -   5. Remove the treated serum from the discs and transfer it to a        suitable storage tube.    -   6. Store the treated serum at 4 EC.

Assay procedure:

-   -   1. Two days before assay, aspirate the storage solution from the        NeutrAvidin^(a) coated white microtiter plate and add 200 μL        plate blocking solution to each well.    -   2. Cover the plate and incubate at ambient temperature for 16-20        hours.    -   3. One day before assay. wash the blocked plate three times with        plate wash buffer, approximately 275 μL per well per wash.        Aspirate the final wash and add 100 μL peptide solution or 100        μL control peptide solution to the appropriate wells of the        plate.    -   4. Cover the plate and incubate with gentle mixing at ambient        temperature for 16-20 hours.    -   5. Day of assay, wash the blocked plate three times with plate        wash buffer, approximately 275 μL per well per wash. Aspirate        the final wash and add 100 μL treated serum to the appropriate        wells of the plate.    -   6. Cover the plate and incubate at 25 EC for 2 hours.    -   7. Wash the blocked plate six times with plate wash buffer,        approximately 275 μL per well per wash. Aspirate the final wash        and add 100 μL conjugate solution to each assay well.    -   8. Cover the plate and incubate at 25 EC for 1.5 hours.    -   9. Wash the blocked plate six times with plate wash buffer,        approximately 275 μL per well per wash. Aspirate the final wash        and add 100 μL substrate solution to each assay well.    -   10. Read the plate at 30 and 60 minutes in a fluorescence        microtiter plate reader set at 365 nm excitation and 450 nm        emission.        Biotinylation of Human Serum Albumin

Materials:

Human Serum Albumin: Sigma A 8763

Sulfosuccinimidyl 6-(biotinamido) Hexanoate: Pierce 21335

Tris base: Sigma T 1503

20 mM sodium phosphate, pH 7.2

100 mM sodium hydroxide solution

Procedure:

Human serum albumin is dissolved in phosphate buffer at a concentrationof approximately 40 mg/mL. The protein concentration of the solution isdetermined by absorbance at 280 nm (1 mg/mL=OD280 of 0.58) or by theLowry method.

Immediately prior to biotinylation, the pH of the albumin solution isadjusted to 8.5 by the addition of sodium hydroxide. Succinimidyl biotinis then added at a molar ratio of 50:1 (422 mg succinimidyl biotin permg albumin). The reaction mixture is vortexed thoroughly and then mixedgently for 45 minutes at ambient temperature.

Reaction byproducts and unreacted biotin are removed by extensivedialysis against phosphate buffer. The biotinylated human serum albuminis stored at 4 C.

Preparation of Covalent Ready Cyanogen Bromide (CNBr) Activated PaperDiscs

Materials:

Paper discs: Schleicher and Schuell 53870

CNBr solution: 20 gm CNBr (Sigma C6388)+600 mL distilled water

1M NaOH

0.05M NaHCO3

25%, 50%, 75%, and 100% acetone

Distilled water

Dessicant packets: Sigma S8394

Zip lock plastic bags

Procedure:

The following procedure is performed under a hooded, well ventilatedenvironment. 20 gm paper discs are swelled in 200 mL distilled water atroom temperature. Swelled paper discs are then added to 600 mL of CNBrsolution while stirring. Bring up the pH of the stirring mixture to 10.5and maintain at pH 10.5 until 100 mL of 1M NaOH have been used up.Aspirate the resulting liquid and wash discs with 500 mL of NaHCO3buffer for 2 minutes at room temperature. Repeat wash step×12. Rinsediscs twice with 500 mL distilled water. Rinse discs twice with 500 mL25% acetone. Rinse discs twice with 500 mL 50% acetone. Rinse discstwice with 500 mL 75% acetone. Rinse discs twice with 500 mL 100%acetone. Aspirate last acetone wash solution and allow discs to dryunder a running fume hood at room temperature. Store dried CNBractivated paper discs in zip lock plastic bags containing dessicantpackettes.

Preparation of Neutravidin Conjugated Paper Discs and Biotinylated HumanSerum Albumin Conjugated Paper Discs

Materials:

Biotinylated human serum albumin: Prepared by method of Example 1

Neutravidin: Pierce 31000

CNBr-activated paper discs: Prepared by method of Example 2

Modified Coca's buffer: 0.05M NaHCO3+0.15M NaCl. PH 7.2

0.05M ethanolamine solution

0.2M sodium acetate buffer, pH 4.0.

Paper disc incubation buffer: 0.05M sodium phosphate+0.1 5M NaCl+0.05%NaN3

+0.5% Tween20

Procedure:

A 2.5 mg/mL solution of neutravidin is prepared in modified Coca'sbuffer. A 2.5 mg/mL solution of biotinylated human serum albumin isprepared in modified Coca's buffer. 50 CNBr-activated discs are added toeach mL of protein solution. Each protein/disc mixture is agitated for16 to 18 hours at room temperature. Each solution surrounding therespective paper discs is aspirated and each set of discs are washed×3with modified Coca's buffer. The washed discs are immersed in 0.05Methanolamine solution and agitated for 3 hours in order to block anyunreacted CNBr sites. Each set of paper discs is then washed×3 with thesodium acetate buffer. During the third step, the paper discs areincubated in the sodium acetate buffer for 30 minutes under gentleagitation. Each set of paper discs is then washed×4 in Coca's buffer andthen stored in the paper disc incubation solution at 4 C.

Preparation of Neutravidin Coated Microtiter Plates

Materials:

Amino Polystyrene Microtiter Plates (White): Nunc 453686 or theequivalent

Neutravidin: Pierce 31000

Disuccinimidyl suberate (DSS): Pierce 21555

Dimethyl sulfoxide (DMSO): Burdick and Jackson 081-1

20 mM sodium phosphate, pH 5.50

50 mM sodium carbonate, pH 9.6

PBS with sodium azide

Procedure:

Prepare a volume of neutravidin appropriate for the number of plates tobe coated. The coating solution contains 20 mg/mL neutravidin in 20 mMsodium phosphate, pH 5.50.

Prepare a suitable volume of DSS, 1.22 mg/mL, in dry DMSO. This solutionmust be used within 2 hours of preparation.

For each plate to be coated, add 60 mL DSS solution to each wellfollowed by 60 mL of 50 mM sodium carbonate, pH 9.6. Incubate thismixture in the wells for 36 minutes at ambient temperature. Aspirate thewells and wash twice with deionized water. Immediately add 100 mLneutravidin solution. Cover the plate and incubate at ambienttemperature for 16-18 hours.

Aspirate the coating solution and add approximately 280 mL PBS withazide to each well. Seal the plate with a foil cover. Store the coatedplates at about 4 C.

Vaccination Methods

Methods for preparing and administering a vaccine using a peptide as anepitope have been reported. For example, Gilewski et al. (2000) used aMUC1 peptide with a keyhole limpet hemocyonin (KLM) in a conjugate todetermine whether an immune response could be generated against the MUCIpeptide that would also bind with tumor cells. An immunogenic responsewas reported.

Passive Immunization Constructs That the Host Immune System Recognizesand Kills Cancer Cells

1. Engineered Antigen Presenting Cells

See the “Detailed Description of the Invention” from U.S. Pat. No.5,851,320, incorporated by reference.

2. Dendritic Cells

See the “Detailed Description of the Invention” and “Examples 1-7” fromU.S. Pat. No. 5,871,156 and the “Detailed Description of the Invention”and “Examples 1-5” from U.S. Pat. No. 6,080,409, incorporated byreference.

Active Immunization Using Cancer Vaccines

1. Recombinant Fusion Proteins

See the “Summary of the Invention” from U.S. Pat. No. 4,399,216 and U.S.Pat. Nos. 6,106,829 and 5,616,477 incorporated by reference.

2. Adjuvants

See U.S. Pat. Nos. 5,750,110; 5,876,966; 5,876,735; 6,013,268 and6,080,399, incorporated by reference.

3. Nucleic Acid Molecules

See U.S. Pat. Nos. 5,593,972; 5,817,637; 5,830,876; 6,063,384;6,077,663; 5,981,505 and 5,942,235 incorporated by reference.

4. Recombinant Microorganisms which Express Cancer Antigens

See U.S. Pat. No. 6,051,237 incorporated by reference.

5. Antibody Delivers Antigen to Antigen Presenting Cells

See U.S. Pat. No. 5,194,254 incorporated by reference.

6. Heat Shock Protein/Antigen Complexes

See U.S. Pat. Nos. 5,837,251; 5,981,706; 5,985,270; 5,997,873; 6,030,618and 6,136,315 incorporated by reference.

7. Cell Lytic Therapeutic Antibodies

See the section entitled “Therapy” in U.S. Pat. No. 4,585,742incorporated by reference.

8. Cell Adhesion Blocking Antibodies Such as Intergrin Antagonists

(See Kerr et al., 2000).

9. Growth Factor Receptor Blocking Antibodies

See U.S. Pat. No. 5,772,997 incorporated by reference.

Presence or Absence of 41 Molecules Listed in Table 3 in Prostate CancerPeptides

Information on the molecules in Table 3 were obtained from the followingsources:

Bryden et al., 1999;

Cress et al., 1995;

Dong et al., 1996;

Fudge et al., 1994;

Giri et al., 1999;

Grasso et al., 1997;

Kimura et al., 1996;

Kramer et al., 1995;

Luo et al., 1999;

Rokhlin et al., 1997;

Takahashi et al., 1998;

Tozawa, 1996;

Tronet. al., 1999;

Watanabe et al., 1999; and

Zheng et al., 2000.

Use of Peptides of the Present Invention on Microchips

Microchips that have oligonucleotides or peptides have been developed bymany groups or researches for various applications e.g. determiningwhether genes are present in a biological sample by determining whetherDNA molecules in the sample hybridize under conditions whereinhybridization implies a specific degree of homology between a DNAmolecule in a sample applied to the microchip and a DNA molecule in themicrochip. Microchips are designed so that questions such as “Is thegene for the disease X present in a person?” or “Does the patient have aparticular mutation?” or “Is there a specific antigen(s) present in thesample?” can be answered by interpreting the hybridization pattern inthe chip, or in the case of antigen or antibody detection, the patternof antigen-antibody complexing on the microchip. Examples of patents inthe microchip area are U.S. Pat. No. 5,861,247 and U.S. Pat. No.5,770,721. Microchips are sold commercially by Affymetrix, Hyseq andother companies. Licenses are available for microchip technologiesthrough Argonne National Laboratory.

Documents Cited

Barratt (1998) J. Immunother 21(2):142-148.

Begum, N. A., et al. (1995) Hepatology. 22(5):1447-1455.

Bogden and Moreau (1998) U.S. Pat. No. 5,736,517.

Bonkhoff, H. (1998), Anal. Quant. Cytol. Histol. 20(5): 437-442.

Boon, T. et al. (1994) U.S. Pat. No. 5,342,774.

Brame, C. J., et al. (1999) J. Biol. Chem. 274(19):13139-13146.

Bryden, et al. (1999) BJU Int. 84(9): 1032-1034.

Calenoff, E. (1998) U.S. Pat. No.5,763,164.

Cheever (1997a) Immunol Rev. 157:177-194.

Cheever (1997b) Adv. Cancer Res. 71:343-371.

Cress, A F et al. (1995) Cancer Metastasis Rev. 1995 14(3):219-228.

Damiano, J. S., et al. (1999) Blood 93(5):1658-1656.

Damjanovich, L., et al. (1997) Acta Chir Hung. 36(1-4):69-71.

DeVita, V T, et al. (1985) Cancer: Principles and Practice of Oncology,2^(nd) edition, Lippincott (Pub).

Disis et al. (1998a) Immunology 93(2):192-199.

Disis et al. (1 998b) Crit Rev Immunol 18(1-2):37-45.

Disis et al. (1999) Clin Cancer Res. 5(6):1289-1297.

Dong, J T, et al. (1996) Cancer Res. 56(19): 4387-4390.

Ekblom, M., et al. (1998) Ann. N.Y. Acad. Sci. 857:194-211.

Fauchere, J. L. and V. Pliska (1983) Eur. J. Med. Chem. 18(4):369-375.

Fleischmajer, R., et al. (1998) Ann. U. U. Acad. Sci. 857:212-227.

Fraga et al. (1990) J. Mol. Recognit. 3(2):65-7347.

Friedl, P., et al. (1998) Cell Adhes. Commun. 6(2-3):225-236.

Fudge, K., et al. (1994) Mod. Pathol. 7(5):549-54.

Fujiwara, H., et al. (1998) Harm. Res. 50 Suppl 2:25t-29.

Furukawa, F., et al. (1994) J. Dermatol. 21(11):802-813.

Gilewski, et al. (2000) Clin. Cancer Res. 6(5):1693-1701.

Giri, D., et al. (1999) Clin. Cancer Res. 5(5):1063-1071.

Goldenberg, D. M. and H. A. Nabi (1999) Semin. Nucl. Med. 29(1):41-48.

Goldenberg, D. M. (1993) Am. J. Med. 94(3):297-312.

Grasso, A W, (1997) Oncogene 15(22):2705-2716.

Henderson et al. (1998) J. Immunother 21(4):247-256.

Hopp et al. (1981) Prox. Natl. Acad Sci. USA. 78(6):3824-3828.

Hudziak (1998a) U.S. Pat. No. 5,772,997.

Hudziak (1998b) Mol. Cell. Biol. 9(3):1165-1172.

Hussain, R., et al. (1996). Biomed. Petp. Proteins Nucleic Acids.2(3):67-70.

Katsura, M., et al. (1998) Gynecol. Oncol. 71(2):185-189.

Kerr et al. (2000) Expert Opin. Investig Drugs 9(6):1271-1279.

Kimura (1966)

Knight et al. U.S. Pat. No. 5,620,955.

Kramer et al. (1995) J. Urol. 154(5) 1636-1641.

Lhansi, F. A., et al. (1998) J. Biol. Chem. 273(12):6928-6936.

Liapis, H., et al. (1996) Diagn. Mol. Pathol. 5(2):127-135.

Lohi, J., et al. (1998) J. Pathol. 184(2):191-196.

Luguki, (1999)

Luo, W., etal. (1999) Cancer Gene Ther. 6(4):313-321.

Parker et al. (1986) Biochemistry. 25:5425-5432.

Rabinovitz, J., et al. (1995) Clin. Exp. Metastasis 13(6):481-491.

Rokhlin et al. (1997) Cancer Res. 57(9) 1758-1768.

Romonov, V. I. and Goligorsky, M. S. (1999) Prostate 39(2):108-118.

Ruoslahti, E. (1997) Kidney Int. 51(5):1413-1417.

Shamsi et al. (1998) Radiology 206(2):365-371.

Suzuki, K. and K. Takahashi (1999). Int. J Oncol. 14(5):897-904.

Takahashi, S. et al. (1998) Cancer Lett 129(1): 97-102.

To, S. Y., et al. (1992) J. Clin. Laser Med. Surg. 10(3):159-169.

Tozawa, K. et al. (1996) Nippon Hinyokika Gakkai Zasshi 87(9):1082-1091.

Tran et al. (1999) Am. J. Pathol. 155(3) 797-798.

Tsimikas, S., et al. (1999) J. Nucl. Cardiol. 6(1Pt1):41-53.

Watanabe, M. et al. (1999) Cancer Lett. 141(1-2):173-178.

Zheng, D. Q., et al. (1999) Cancer Res. 59(7):1655-1664.

U.S. Pat. No. 4,399,216.

U.S. Pat. No. 4,585,742.

U.S. Pat. No. 5,194,254.

U.S. Pat. No. 5,593,972.

U.S. Pat. No. 5,616,477.

U.S. Pat. No. 5,750,110.

U.S. Pat. No. 5,770,721.

U.S. Pat. No. 5,817,637.

U.S. Pat. No. 5,830,876.

U.S. Pat. No. 5,837,251.

U.S. Pat. No. 5,861,247.

U.S. Pat. No. 5,871,756.

U.S. Pat. No. 5,876,735.

U.S. Pat. No. 5,876,966.

U.S. Pat. No. 5,942,235.

U.S. Pat. No. 5,962,320.

U.S. Pat. No. 5,981,505.

U.S. Pat. No. 5,981,706.

U.S. Pat. No. 5,985,270.

U.S. Pat. No. 5,997,873.

U.S. Pat. No. 6,013,268.

U.S. Pat. No. 6,030,618.

U.S. Pat. No. 6,051,237.

U.S. Pat. No. 6,063,384.

U.S. Pat. No. 6,077,663.

U.S. Pat. No. 6,080,399.

U.S. Pat. No. 6,080,409.

U.S. Pat. No. 6,106,829.

U.S. Pat. No. 6,136,315.

1. (canceled)
 2. A cancer-specific or highly cancer-associated peptidecomprising the following structure: (a) an amino acid sequence of alength from 3-1000 amino acids; (b) a net hydrophilic character; and (c)at least one glycosylatable amino acid located at a position in theamino acid sequence no further than 3 amino acids away from the aminoacid adjacent to either end of the peptide, wherein for cells withnormal growth patterns the amino acid is the site of glycosylation, butin cancer cells the site is missing entirely, so that the glycosylationsite confers a cancer-specific or highly cancer-associatedimmunogenicity or marker function to the peptide.
 3. The peptide ofclaim 2 further defined as immunogenic.
 4. The peptide of claim 2,wherein the glycosylatable amino acid is asparagine.
 5. The peptide ofclaim 2, further comprising a plurality of deglycosylated amino acids,and wherein each deglycosylated amino acid is separated from thedeglycosylated amino acid nearest to it by no more than 6 unmodifiedamino acids.
 6. The peptide of claim 5 further comprising: (a) achemical modification of at least one of the deglycosylated amino acidswherein the chemical modification confers upon the peptide an additionalcancer-specific or highly cancer-associated immunogenicity than that dueto glycosylation; and (b) an amino acid sequence wherein no more than 3unmodified amino acids are located on either side of a modified aminoacid or amino acid that has a glycosylation site removed.
 7. (canceled).8. The peptide of claim 2, wherein the peptide is producedsynthetically.
 9. The peptide of claim 2, produced by the method ofclaim
 1. 10. A composition comprising a peptide of claim
 2. 11. Animmunogenic composition capable of inducing a mammal to produceantibodies specific for an epitope on a cancer cell, wherein theimmunogenic composition comprises a peptide of claim
 2. 12-23.(canceled)
 24. A therapeutic construct comprising a peptide of claim 2and (a) adjuvant/peptide conjugates comprising the peptide coupled tomolecule which facilitates enhanced immunogenicity; and (b) neomoleculescreated by recombinant techniques containing a peptide with adjuvantmolecular sequences which promote increased immunogenicity of thepeptide of claim
 2. 25-26. (canceled)
 27. The peptide of claim 2 havinga sequence selected from the group consisting of: uNu uNuu Nuuu uNuuuuuNuu uuNuuu uuuNuuu uNuMuuu uuNuMuuu uuuNuMuuu uuuNMMuuu uuuNuMMuuuuuuNMMMuuu uNuuMuu uNuuMuuu uuNuuMuuu uuuNuuMuuu uuNuuMuuNuuuuuNuuMuuNuuu uuuMuuNuuMuuu [uuNuuN]n [uuuNuuuN]n [uuuNuuuuN]n[uuuNuuuuuN]n [uuuNuuuuuN]n [uuuNuuuuuuN]n [uuuNuuuuuuNu]n[uuuNuuuuuuNuu]n [uuuNuuuuuuNuuu]n [uuMuuN]n

wherein u is an unmodified amino acid, N is a deglycosylated amino acid,and M is a modified amino acid.